Copper-associated liver disease (Proceedings)

Copper is an essential trace element in diets and is required for a number of physiologically important enzymes. Cells have highly specialized and complex systems for maintaining intracellular copper concentrations. At toxic concentrations, free intracellular copper initiates oxidative damage causing hepatocellular necrosis and inflammation. Copper accumulation in the liver can be associated with significant hepatic injury resulting in acute hepatitis, chronic hepatitis, and cirrhosis. It is one of the few well-documented causes of canine chronic hepatitis. In one study, copper-associated hepatitis (acute and chronic) accounted for 1/3 of all dogs with primary hepatitis. Hepatic copper accumulation and hepatopathy have been described in cats but appears to be rare. The severity of hepatic injury correlates with the amount of hepatic copper, but subcellular localization of molecules and the molecular association also plays a role. Serum copper levels do not accurately reflect hepatic copper content and quantitative analysis of copper in the liver is required. Hepatic copper concentration in normal dogs is between 150 - 400 ugm/gm dry weight (ppm). Inflammatory hepatic injury does not consistently occur until copper concentrations exceed 2,000 ugm/gm dry weight. However, there may be breed variations; for example, in Doberman pinschers hepatic inflammation is present with lower copper concentrations. Transient acquired Fanconi's syndrome has been described in dogs with excess hepatic copper accumulation. Copper granules were demonstrated on renal biopsy in some but not all dogs.

Potential mechanisms for hepatic copper accumulation include primary metabolic defects in hepatic copper metabolism, cholestasis causing impaired biliary excretion of copper, and excess copper absorption. A primary defect in hepatic copper metabolism occurs in Bedlington terriers with a genetic mutation in the gene encoding the copper transport protein, COMMD1 (formerly MURR1), resulting in a defect in biliary copper excretion. In the early stages, copper is sequestered in hepatic lysosomes and hepatic damage is minimal. However, with progressive accumulation of copper, hepatic injury becomes significant. The average copper concentration in Bedlington terriers with chronic hepatitis is approximately 6,000 ugm/gm dry weight and values up to 12,000 ugm/gm dry weight have been reported. Inherited copper-associated liver disease is also described in the West Highland white terrier, Skye terrier, Doberman pinscher, Dalmatian, and Labrador retriever, but with the possible exception of Dalmatians, the hepatic copper levels are much lower than in Bedlington terriers. The pathogenesis of copper accumulation and the relationship to chronic liver disease in these breeds is poorly understood. It seems likely that these breeds have a hereditary disorder of copper handling, but it is unlikely to be the same as described for the Bedlington terrier. Breed related disorders are discussed in more detail in a subsequent section of this chapter.

Hepatic copper accumulation in the liver may also be a consequence rather than the cause of chronic hepatitis. Since copper is normally excreted in the bile, chronic cholestasis and impaired bile flow can result in secondary copper accumulation. Secondary copper accumulation is predominantly periportal and is usually less than 2000 ugm/gm dry weight. The effect of cholestasis on hepatic copper content was evaluated in three groups of dogs: Bedlington terriers with copper toxicity, dogs with extrahepatic biliary obstruction (the prototype example of a cholestatic disorder) and chronic hepatitis in breeds not known to be at risk for copper-associated liver disease. Hepatic copper content was evaluated by a semi-quantitative method based on copper staining of liver tissue with rubeanic acid, using a scale of 0 (no copper) to five. Copper staining revealed absent to mild increases (scores of 0-2+) in dogs with biliary obstruction and chronic hepatitis when compared to Bedlington Terriers (scores of 5+). It was concluded that copper scores of 3+ or higher were suggestive of a primary copper storage disease. Unfortunately, quantitative copper analysis was not evaluated. Markers of oxidative injury and altered defense mechanisms were similar in the 3 groups, consistent with the concept that copper, inflammation, and cholestasis can all contribute to oxidative injury.

Many other breeds of dogs (including mixed breeds) have been identified with increased hepatic copper and chronic hepatic disease but a hereditary mechanism has not been proven. When liver disease and copper accumulation are identified in a breed of dog not previously described with familial copper-associated disorder, it can be difficult to determine whether copper accumulation is primary or secondary. Findings that would support a primary metabolic defect in copper metabolism include copper accumulation that precedes cholestasis or inflammation, centrolobular (zone 3) distribution of copper, histochemical score for copper of 3+ or greater, or quantitative copper measurements that exceed 2000 ugm/gm dry weight.

High dietary copper intake appears to be an uncommon cause of hepatic copper accumulation, although it was suspected in two farm dogs chronically eating commercial calf food supplemented with copper. The copper content of commercial dog foods range from 12-16 mg/kg dry matter, which is relatively high compared to recommended minimum daily copper requirements in dogs. Commercial dog food alone does not appear to explain hepatic copper accumulation and liver disease in dogs. However, it has been speculated that the recent increase in pathologically elevated hepatic copper concentrations (specifically evaluated in Labrador retrievers), may coincide with a pet food industry recommendation to replace cupric/cuprous oxide in feed formulations because of its low bioavailability.

On H&E staining, excess copper appears as golden brown refractile granules. Histochemical stains, such as rhodanine or rubeanic acid, can be used to semi-quantitatively evaluate for copper in the liver. These stains consistently detect copper when amounts exceed 400 ugm/gm dry weight. Values obtained by quantitative copper analysis have a strong correlation with the number and size of granules seen with histochemical stains within the range of 400 to 1000 ugm copper/gm of liver tissue. Zonal distribution of copper accumulation should be noted, since copper accumulation starting in the centrolobular area is more likely with a primary metabolic defect in copper metabolism. Copper granules can also be detected on cytology of hepatic aspirates or impression smears stained with rhodanine or rubeanic acid. Quantitative analysis for copper, by atomic absorption analysis on fresh hepatic tissue, is the definitive method to document increased hepatic copper content. Approximately 1 gram of tissue is required, which should be shipped frozen or refrigerated to the laboratory in a copper-free serum blood tube. Needle core biopsy specimens may not be reliable for metal analysis, since copper and iron values are consistently lower in needle core versus wedge biopsy samples. Formalin-fixed tissues should be avoided, since formalin may contain copper or leach copper from the tissue. Hepatic copper can be reliably determined retrospectively on deparaffinized-archived liver biopsy specimens. Many dogs with copper-associated chronic hepatitis also have increased hepatic iron concentrations. Hepatic iron accumulation usually correlates with degree of inflammation. Whether iron, as an oxidant, interacts with copper to contribute to lesions seen in copper-associated hepatitis remains to be seen.

Dogs with hepatic copper concentrations > 1500 ugm/gm, should be treated with the copper chelator, penicillamine (10-15 mg/kg PO q 12 hours). Treatment usually requires months to years to produce significant decreases in hepatic copper. A mean decrease in copper of approximately 1,500 ugm/gm was achieved in Bedlington terriers treated for 6 months. Dogs with secondary copper accumulation appear to respond more rapidly, possibly because of the lower hepatic copper content. Doberman pinschers with subclinical hepatitis treated with penicillamine for 4 months had a mean decrease in copper from 1036 ugm/gm to 407 ugm/gm. Penicillamine has additional effects beyond copper chelation, which may be beneficial in dogs with chronic hepatitis including inhibition of collagen deposition, stimulation of collagenase activity, immunosuppression, and immunomodulation. Common side effects of penicillamine therapy include anorexia, nausea, and vomiting, which can be minimized by giving the medication with a small amount of food. The copper chelator, trientine (10-15 mg/kg PO q 12 hours), is also effective for reducing hepatic copper concentrations. It has fewer side effects than penicillamine and is effective in dogs with hemolytic anemia due to copper release from necrotic hepatocytes. Copper deficiency (microcytosis and hepatic dysfunction) has been described in a dog treated with long-term copper chelator therapy (trientine) and a copper restricted diet. Decisions on duration of chelator therapy are based on follow-up liver biopsies with periodic monitoring of quantitative hepatic copper content. Lifelong therapy may be required.

Oral zinc salts can be used for maintenance therapy after copper chelation, or as initial therapy in dogs with hepatic copper concentrations between 400 ug/g dry and 1500 ug/g dry weight. Zinc supplementation is typically used in conjunction with dietary copper restriction. Zinc decreases intestinal copper absorption by inducing the intestinal copper-binding protein, metallothionein, within intestinal epithelial cells, which preferentially binds dietary copper and prevents its absorption. Zinc acetate is given at a dose of 100 mg PO BID for 2-3 months, then at a maintenance dose of 50 mg PO BID. A minimum of 3 months of zinc therapy is required before copper uptake from the intestinal tract is blocked. Zinc administration should be separated from meals by at least 1 hour and should theoretically not be given at the same time as a copper chelator. Serum zinc concentrations should be monitored to achieve a level of 200-400 ug/dl. Zinc concentrations greater than 500 ug/dl may be toxic (hemolytic anemia).

Low copper diets are most beneficial for managing early (subclinical) copper accumulation in dogs affected with primary metabolic defects in hepatic copper metabolism.

Feeding a low copper diet has been shown to decrease hepatic copper content in Labrador retrievers with subclinical copper-associated liver disease. Additional treatment with zinc did not appear to increase the copper-lowering effect of dietary management. Foods containing large amounts of copper (liver, other organ meats, shellfish, eggs, bean/legumes, chocolate, nuts, cereals, and copper-containing vitamin supplements) should be avoided.

Since oxidative stress is a significant mechanism for hepatic damage associated with copper accumulation, antioxidant therapy with Vitamin E (10-15 IU/kg/day), or S-adenosylmethionine (SAMe)(20 mg/kg/day) would seem reasonable. Other cytoprotective agents such as silymarin (milk thistle) and ursodeoxycholic acid might also be beneficial.

Suggested Reading: Hoffmann G. Vet Clin North Am: Small Anim 39:489-511, 2009.